Treg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke.

The precise mechanisms underlying the beneficial effects of regulatory T (Treg) cells on long-term tissue repair remain elusive. Here, using single-cell RNA sequencing and flow cytometry, we found that Treg cells infiltrated the brain 1 to 5 weeks after experimental stroke in mice. Selective depletion of Treg cells diminished oligodendrogenesis, white matter repair, and functional recovery after stroke. Transcriptomic analyses revealed potent immunomodulatory effects of brain-infiltrating Treg cells on other immune cells, including monocyte-lineage cells. Microglia depletion, but not T cell lymphopenia, mitigated the beneficial effects of transferred Treg cells on white matter regeneration. Mechanistically, Treg cell-derived osteopontin acted through integrin receptors on microglia to enhance microglial reparative activity, consequently promoting oligodendrogenesis and white matter repair. Increasing Treg cell numbers by delivering IL-2:IL-2 antibody complexes after stroke improved white matter integrity and rescued neurological functions over the long term. These findings reveal Treg cells as a neurorestorative target for stroke recovery.

Treg Cells Promote the SREBP1-Dependent Metabolic Fitness of Tumor-Promoting Macrophages via Repression of CD8+ T Cell-Derived Interferon-γ.

Regulatory T (Treg) cells are crucial for immune homeostasis, but they also contribute to tumor immune evasion by promoting a suppressive tumor microenvironment (TME). Mice with Treg cell-restricted Neuropilin-1 deficiency show tumor resistance while maintaining peripheral immune homeostasis, thereby providing a controlled system to interrogate the impact of intratumoral Treg cells on the TME. Using this and other genetic models, we showed that Treg cells shaped the transcriptional landscape across multiple tumor-infiltrating immune cell types. Treg cells suppressed CD8+ T cell secretion of interferon-γ (IFNγ), which would otherwise block the activation of sterol regulatory element-binding protein 1 (SREBP1)-mediated fatty acid synthesis in immunosuppressive (M2-like) tumor-associated macrophages (TAMs). Thus, Treg cells indirectly but selectively sustained M2-like TAM metabolic fitness, mitochondrial integrity, and survival. SREBP1 inhibition augmented the efficacy of immune checkpoint blockade, suggesting that targeting Treg cells or their modulation of lipid metabolism in M2-like TAMs could improve cancer immunotherapy.

Insulin secretion deficits in a Prader-Willi syndrome ß-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones.

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in ß-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS ß-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS ß-cells. Consistent with reduced ER chaperones levels, PWS INS-1 ß-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS ß-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic ß-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and ß-cell secretory pathway function.

The CHARGE syndrome ortholog CHD-7 regulates TGF-ß pathways in Caenorhabditis elegans.

CHARGE syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding protein-7 (CHD7) and characterized by retarded growth and malformations in the heart and nervous system. Despite the public health relevance of this disorder, relevant cellular pathways and targets of CHD7 that relate to disease pathology are still poorly understood. Here we report that chd-7, the nematode ortholog of Chd7, is required for dauer morphogenesis, lifespan determination, stress response, and body size determination. Consistent with our discoveries, we found chd-7 to be allelic to scd-3, a previously identified dauer suppressor from the DAF-7/ tumor growth factor-ß (TGF-ß) pathway. Epistatic analysis places CHD-7 at the level of the DAF-3/DAF-5 complex, but we found that CHD-7 also directly impacts the expression of multiple components of this pathway. Transcriptomic analysis revealed that chd-7 mutants fail to repress daf-9 for execution of the dauer program. In addition, CHD-7 regulates the DBL-1/BMP pathway components and shares roles in male tail development and cuticle synthesis. To explore a potential conserved function for chd-7 in vertebrates, we used Xenopus laevis embryos, an established model to study craniofacial development. Morpholino-mediated knockdown of Chd7 led to a reduction in col2a1 messenger RNA (mRNA) levels, a collagen whose expression depends on TGF-ß signaling. Both embryonic lethality and craniofacial defects in Chd7-depleted tadpoles were partially rescued by overexpression of col2a1 mRNA. We suggest that Chd7 has conserved roles in regulation of the TGF-ß signaling pathway and pathogenic Chd7 could lead to a defective extracellular matrix deposition.

In vivo evolution of a Klebsiella pneumoniae capsule defect with wcaJ mutation promotes complement-mediated opsono-phagocytosis during recurrent infection.

BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct ELISA, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In five genetically-related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsono-phagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsono-phagocytosis, which may promote KPC-Kp persistence by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

A genetically inducible endothelial niche enables vascularization of human kidney organoids with multilineage maturation and emergence of renin expressing cells.

Vascularization plays a critical role in organ maturation and cell-type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.

iPSC-Derived Neurons from Patients with POLG Mutations Exhibit Decreased Mitochondrial Content and Dendrite Simplification.

Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase gamma, result in clinical syndromes characterized by mitochondrial DNA (mtDNA) depletion in affected tissues with variable organ involvement. The brain is one of the most affected organs, and symptoms include intractable seizures, developmental delay, dementia, and ataxia. Patient-derived induced pluripotent stem cells (iPSCs) provide opportunities to explore mechanisms in affected cell types and potential therapeutic strategies. Fibroblasts from two patients were reprogrammed to create new iPSC models of POLG-related mitochondrial diseases. Compared with iPSC-derived control neurons, mtDNA depletion was observed upon differentiation of the POLG-mutated lines to cortical neurons. POLG-mutated neurons exhibited neurite simplification with decreased mitochondrial content, abnormal mitochondrial structure and function, and increased cell death. Expression of the mitochondrial kinase PTEN-induced kinase 1 (PINK1) mRNA was decreased in patient neurons. Overexpression of PINK1 increased mitochondrial content and ATP:ADP ratios in neurites, decreasing cell death and rescuing neuritic complexity. These data indicate an intersection of polymerase gamma and PINK1 pathways that may offer a novel therapeutic option for patients affected by this spectrum of disorders.

Non-canonical Wnt/calcium signaling is protective against podocyte injury and glomerulosclerosis.

Activation of canonical Wnt signaling has been implicated in podocyte injury and proteinuria. As Wnts are secreted proteins, whether Wnts derived from podocytes are obligatory for promoting proteinuria remains unknown. To address this, we generated conditional knockout mice where Wntless, a cargo receptor protein required for Wnt secretion, was specifically deleted in glomerular podocytes. Mice with podocyte-specific ablation of Wntless (Podo-Wntless-/-) were phenotypically normal. However, after inducing kidney damage with Adriamycin for six days, Podo-Wntless-/- mice developed more severe podocyte injury and albuminuria than their control littermates. Surprisingly, ablation of Wntless resulted in upregulation of ß-catenin, accompanied by reduction of nephrin, podocin, podocalyxin, and Wilms tumor 1 proteins. In chronic injury induced by Adriamycin, increased albuminuria, aggravated podocyte lesions and extracellular matrix deposition were evident in Podo-Wntlessl-/- mice, compared to wild type mice. Mechanistically, specific ablation of Wntless in podocytes caused down-regulation of the nuclear factor of activated T cell 1 (NFAT1) and Nemo-like kinase (NLK), key downstream mediators of non-canonical Wnt/calcium signaling. In vitro, knockdown of either NFAT1 or NLK induced ß-catenin activation while overexpression of NLK significantly repressed ß-catenin induction and largely preserved nephrin in glomerular podocytes. Thus, our results indicate that podocyte-derived Wnts play an important role in protecting podocytes from injury by repressing ß-catenin via activating non-canonical Wnt/calcium signaling.

Loss of Anks6 leads to YAP deficiency and liver abnormalities.

ANKS6 is a ciliary protein that localizes to the proximal compartment of the primary cilium, where it regulates signaling. Mutations in the ANKS6 gene cause multiorgan ciliopathies in humans, which include laterality defects of the visceral organs, renal cysts as part of nephronophthisis and congenital hepatic fibrosis (CHF) in the liver. Although CHF together with liver ductal plate malformations are common features of several human ciliopathy syndromes, including nephronophthisis-related ciliopathies, the mechanism by which mutations in ciliary genes lead to bile duct developmental abnormalities is not understood. Here, we generated a knockout mouse model of Anks6 and show that ANKS6 function is required for bile duct morphogenesis and cholangiocyte differentiation. The loss of Anks6 causes ciliary abnormalities, ductal plate remodeling defects and periportal fibrosis in the liver. Our expression studies and biochemical analyses show that biliary abnormalities in Anks6-deficient livers result from the dysregulation of YAP transcriptional activity in the bile duct-lining epithelial cells. Mechanistically, our studies suggest, that ANKS6 antagonizes Hippo signaling in the liver during bile duct development by binding to Hippo pathway effector proteins YAP1, TAZ and TEAD4 and promoting their transcriptional activity. Together, this study reveals a novel function for ANKS6 in regulating Hippo signaling during organogenesis and provides mechanistic insights into the regulatory network controlling bile duct differentiation and morphogenesis during liver development.

Intestinal Sulfation Is Essential to Protect Against Colitis and Colonic Carcinogenesis.

BACKGROUND & AIMS: Sulfation is a conjugation reaction essential for numerous biochemical and cellular functions in mammals. The 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the key enzyme to generate PAPS, which is the universal sulfonate donor for all sulfation reactions. The goal of this study was to determine whether and how PAPSS2 plays a role in colitis and colonic carcinogenesis. METHODS: Tissue arrays of human colon cancer specimens, gene expression data, and clinical features of cancer patients were analyzed. Intestinal-specific Papss2 knockout mice (Papss2ΔIE) were created and subjected to dextran sodium sulfate-induced colitis and colonic carcinogenesis induced by a combined treatment of azoxymethane and dextran sodium sulfate or azoxymethane alone. RESULTS: The expression of PAPSS2 is decreased in the colon cancers of mice and humans. The lower expression of PAPSS2 in colon cancer patients is correlated with worse survival. Papss2ΔIE mice showed heightened sensitivity to colitis and colon cancer by damaging the intestinal mucosal barrier, increasing intestinal permeability and bacteria infiltration, and worsening the intestinal tumor microenvironment. Mechanistically, the Papss2ΔIE mice exhibited reduced intestinal sulfomucin content. Metabolomic analyses revealed the accumulation of bile acids, including the Farnesoid X receptor antagonist bile acid tauro-ß-muricholic acid, and deficiency in the formation of bile acid sulfates in the colon of Papss2ΔIE mice. CONCLUSIONS: We have uncovered an important role of PAPSS2-mediated sulfation in colitis and colonic carcinogenesis. Intestinal sulfation may represent a potential diagnostic marker and PAPSS2 may serve as a potential therapeutic target for inflammatory bowel disease and colon cancer.

Concentric organization of A- and B-type lamins predicts their distinct roles in the spatial organization and stability of the nuclear lamina.

The nuclear lamina is an intermediate filament meshwork adjacent to the inner nuclear membrane (INM) that plays a critical role in maintaining nuclear shape and regulating gene expression through chromatin interactions. Studies have demonstrated that A- and B-type lamins, the filamentous proteins that make up the nuclear lamina, form independent but interacting networks. However, whether these lamin subtypes exhibit a distinct spatial organization or whether their organization has any functional consequences is unknown. Using stochastic optical reconstruction microscopy (STORM) our studies reveal that lamin B1 and lamin A/C form concentric but overlapping networks, with lamin B1 forming the outer concentric ring located adjacent to the INM. The more peripheral localization of lamin B1 is mediated by its carboxyl-terminal farnesyl group. Lamin B1 localization is also curvature- and strain-dependent, while the localization of lamin A/C is not. We also show that lamin B1's outer-facing localization stabilizes nuclear shape by restraining outward protrusions of the lamin A/C network. These two findings, that lamin B1 forms an outer concentric ring and that its localization is energy-dependent, are significant as they suggest a distinct model for the nuclear lamina-one that is able to predict its behavior and clarifies the distinct roles of individual nuclear lamin proteins and the consequences of their perturbation.

Genetic or pharmacologic Nrf2 activation increases proteinuria in chronic kidney disease in mice.

The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway upregulates key cellular defenses. Clinical trials are utilizing pharmacologic Nrf2 inducers such as bardoxolone methyl to treat chronic kidney disease, but Nrf2 activation has been linked to a paradoxical increase in proteinuria. To understand this effect, we examined genetically engineered mice with elevated Nrf2 signaling due to reduced expression of the Nrf2 inhibitor, Kelch-like ECH-associated protein 1 (Keap1). These Keap1FA/FA mice lacked baseline proteinuria but exhibited increased proteinuria in experimental models evoked by adriamycin, angiotensin II, or protein overload. After injury, Keap1FA/FA mice had increased glomerulosclerosis, nephrin disruption and shedding, podocyte injury, foot process effacement, and interstitial fibrosis. Keap1FA/FA mice also had higher daytime blood pressures and lower heart rates measured by radiotelemetry. Conversely, Nrf2 knockout mice were protected from proteinuria. We also examined the pharmacologic Nrf2 inducer CDDO-Im. Compared to angiotensin II alone, the combination of angiotensin II and CDDO-Im significantly increased proteinuria, a phenomenon not observed in Nrf2 knockout mice. This effect was not accompanied by additional increases in blood pressure. Finally, Nrf2 was found to be upregulated in the glomeruli of patients with focal segmental glomerulosclerosis, diabetic nephropathy, fibrillary glomerulonephritis, and membranous nephropathy. Thus, our studies demonstrate that Nrf2 induction in mice may exacerbate proteinuria in chronic kidney disease.

The N-cadherin interactome in primary cardiomyocytes as defined using quantitative proximity proteomics.

The junctional complexes that couple cardiomyocytes must transmit the mechanical forces of contraction while maintaining adhesive homeostasis. The adherens junction (AJ) connects the actomyosin networks of neighboring cardiomyocytes and is required for proper heart function. Yet little is known about the molecular composition of the cardiomyocyte AJ or how it is organized to function under mechanical load. Here, we define the architecture, dynamics and proteome of the cardiomyocyte AJ. Mouse neonatal cardiomyocytes assemble stable AJs along intercellular contacts with organizational and structural hallmarks similar to mature contacts. We combine quantitative mass spectrometry with proximity labeling to identify the N-cadherin (CDH2) interactome. We define over 350 proteins in this interactome, nearly 200 of which are unique to CDH2 and not part of the E-cadherin (CDH1) interactome. CDH2-specific interactors comprise primarily adaptor and adhesion proteins that promote junction specialization. Our results provide novel insight into the cardiomyocyte AJ and offer a proteomic atlas for defining the molecular complexes that regulate cardiomyocyte intercellular adhesion. This article has an associated First Person interview with the first authors of the paper.

Growth and mineralization of osteoblasts from mesenchymal stem cells on microporous membranes: Epithelial-like growth with transmembrane resistance and pH gradient.

Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 µm pores at a density of (2 ± 0.4) x 106 pores per cm2. Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was ∼1400 Ω•cm2 at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production.

PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors.

Necroptosis, a form of regulated necrotic cell death, is governed by RIP1/RIP3-mediated activation of MLKL. However, the signaling process leading to necroptotic death remains to be elucidated. In this study, we found that PUMA, a proapoptotic BH3-only Bcl-2 family member, is transcriptionally activated in an RIP3/MLKL-dependent manner following induction of necroptosis. The induction of PUMA, which is mediated by autocrine TNF-α and enhanced NF-κB activity, contributes to necroptotic death in RIP3-expressing cells with caspases inhibited. On induction, PUMA promotes the cytosolic release of mitochondrial DNA and activation of the DNA sensors DAI/Zbp1 and STING, leading to enhanced RIP3 and MLKL phosphorylation in a positive feedback loop. Furthermore, deletion of PUMA partially rescues necroptosis-mediated developmental defects in FADD-deficient embryos. Collectively, our results reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptotic death in vitro and in vivo.

Nitrite attenuates mitochondrial impairment and vascular permeability induced by ischemia-reperfusion injury in the lung.

Primary graft dysfunction (PGD) is directly related to ischemia-reperfusion (I/R) injury and a major obstacle in lung transplantation (LTx). Nitrite (NO2-), which is reduced in vivo to form nitric oxide (NO), has recently emerged as an intrinsic signaling molecule with a prominent role in cytoprotection against I/R injury. Using a murine model, we provide the evidence that nitrite mitigated I/R-induced injury by diminishing infiltration of immune cells in the alveolar space, reducing pulmonary edema, and improving pulmonary function. Ultrastructural studies support severe mitochondrial impairment in the lung undergoing I/R injury, which was significantly protected by nitrite treatment. Nitrite also abrogated the increased pulmonary vascular permeability caused by I/R. In vitro, hypoxia-reoxygenation (H/R) exacerbated cell death in lung epithelial and microvascular endothelial cells. This contributed to mitochondrial dysfunction as characterized by diminished complex I activity and mitochondrial membrane potential but increased mitochondrial reactive oxygen species (mtROS). Pretreatment of cells with nitrite robustly attenuated mtROS production through modulation of complex I activity. These findings illustrate a potential novel mechanism in which nitrite protects the lung against I/R injury by regulating mitochondrial bioenergetics and vascular permeability.

A Model of Dormant-Emergent Metastatic Breast Cancer Progression Enabling Exploration of Biomarker Signatures.

Breast cancer mortality predominantly results from dormant micrometastases that emerge as fatal outgrowths years after initial diagnosis. In order to gain insights concerning factors associated with emergence of liver metastases, we recreated spontaneous dormancy in an all-human ex vivo hepatic microphysiological system (MPS). Seeding this MPS with small numbers (<0.05% by cell count) of the aggressive MDA-MB-231 breast cancer cell line, two populations formed: actively proliferating ("growing"; EdU+), and spontaneously quiescent ("dormant"; EdU-). Following treatment with a clinically standard chemotherapeutic, the proliferating cells were eliminated and only quiescent cells remained; this residual dormant population could then be induced to a proliferative state ("emergent"; EdU+) by physiologically-relevant inflammatory stimuli, lipopolysaccharide (LPS) and epidermal growth factor (EGF). Multiplexed proteomic analysis of the MPS effluent enabled elucidation of key factors and processes that correlated with the various tumor cell states, and candidate biomarkers for actively proliferating (either primary or secondary emergence) versus dormant metastatic cells in liver tissue. Dormancy was found to be associated with signaling reflective of cellular quiescence even more strongly than the original tumor-free liver tissue, whereas proliferative nodules presented inflammatory signatures. Given the minimal tumor burden, these markers likely represent changes in the tumor microenvironment rather than in the tumor cells. A computational decision tree algorithm applied to these signatures indicated the potential of this MPS for clinical discernment of each metastatic stage from blood protein analysis.

Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.

G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT1), its associated G protein, and ß-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT1 signal-transduction pathway inhibiting stress-mediated cytochrome c release and caspase activation. These findings coupled with our observation that mitochondrial MT1 overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, "automitocrine," analogous to "autocrine" when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand-receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.

E-cadherin is downregulated in benign prostatic hyperplasia and required for tight junction formation and permeability barrier in the prostatic epithelial cell monolayer.

BACKGROUND: We previously reported the presence of prostate-specific antigen (PSA) in the stromal compartment of benign prostatic hyperplasia (BPH). Since PSA is expressed exclusively by prostatic luminal epithelial cells, PSA in the BPH stroma suggests increased tissue permeability and the compromise of epithelial barrier integrity. E-cadherin, an important adherens junction component and tight junction regulator, is known to exhibit downregulation in BPH. These observations suggest that the prostate epithelial barrier is disrupted in BPH and E-cadherin downregulation may increase epithelial barrier permeability. METHODS: The ultra-structure of cellular junctions in BPH specimens was observed using transmission electron microscopy (TEM) and E-cadherin immunostaining analysis was performed on BPH and normal adjacent specimens from BPH patients. In vitro cell line studies using benign prostatic epithelial cell lines were performed to determine the impact of small interfering RNA knockdown of E-cadherin on transepithelial electrical resistance and diffusion of fluorescein isothiocyanate (FITC)-dextran in transwell assays. RESULTS: The number of kiss points in tight junctions was reduced in BPH epithelial cells as compared with the normal adjacent prostate. Immunostaining confirmed E-cadherin downregulation and revealed a discontinuous E-cadherin staining pattern in BPH specimens. E-cadherin knockdown increased monolayer permeability and disrupted tight junction formation without affecting cell density. CONCLUSIONS: Our results indicate that tight junctions are compromised in BPH and loss of E-cadherin is potentially an important underlying mechanism, suggesting targeting E-cadherin loss could be a potential approach to prevent or treat BPH.

Interferon regulatory factor 1-Rab27a regulated extracellular vesicles promote liver ischemia/reperfusion injury.

The role and regulators of extracellular vesicle (EV) secretion in hepatic ischemia/reperfusion (IR) injury have not been defined. Rab27a is a guanosine triphosphatase known to control EV release. Interferon regulatory factor 1 (IRF-1) is a transcription factor that plays an important role in liver IR and regulates certain guanosine triphosphatases. However, the relationships among IRF-1, Rab27a, and EV secretion are largely unknown. Here, we show induction of IRF-1 and Rab27a both in vitro in hypoxic hepatocytes and in vivo in warm IR and orthotopic liver transplantation livers. Interferon γ stimulation, IRF-1 transduction, or IR promoted Rab27a expression and EV secretion. Meanwhile, silencing of IRF-1 decreased Rab27a expression and EV secretion. Rab27a silencing decreased EV secretion and liver IR injury. Ten putative IRF-1 binding motifs in the 1,692-bp Rab27a promoter region were identified. Chromatin immunoprecipitation and electrophoretic mobility shift assay verified five functional IRF-1 binding motifs, which were confirmed by a Rab27a promoter luciferase assay. IR-induced EVs contained higher oxidized phospholipids (OxPL). OxPLs on the EV surface activated neutrophils through the toll-like receptor 4 pathway. OxPL-neutralizing E06 antibody blocked the effect of EVs and decreased liver IR injury. CONCLUSION: These findings provide a novel mechanism by which IRF-1 regulates Rab27a transcription and EV secretion, leading to OxPL activation of neutrophils and subsequent hepatic IR injury. (Hepatology 2018;67:1056-1070).